This application is being submitted to PA-18-591 in accordance with NOT-OD-18-195. Children with Down syndrome (DS) have an approximately 20-fold increased risk of developing acute lymphoblastic leukemia (ALL), and children with DS-ALL have higher rates of treatment-related mortality and rates of relapse. Determining the genetic and environmental modifiers of ALL risk in children with DS will be imperative for future risk stratification and disease prevention efforts in the DS population, and may also inform the etiology of ALL in non-DS individuals. This proposal aims to examine whether children with DS who develop ALL demonstrate differences in DNA methylation and/or patterns of immune development at birth compared with DS children who do not develop leukemia. Previous studies have identified genome-wide epigenetic effects of trisomy 21; however, this has not been comprehensively examined in epidemiologic studies of DS-ALL. Children with DS are born with dysregulated immune systems, presenting with lymphocytopenia of both B-cells and T-cells, and DS individuals also produce higher levels of pro-inflammatory cytokines. The role of neonatal immune development in casecontrol studies of DS-ALL has yet to be performed, thus it is not known whether DS children who go on to develop ALL present with different neonatal blood cell proportions and/or biomarkers of immune development at birth compared with DS children who do not develop leukemia. These are exactly the goals of the parent project for which this supplement request is based upon, albeit for the genesis of ALL in non-DS children. In this study, which includes a total of 190 DS-ALL cases and 290 DS non-leukemia controls, we aim: Aim 1: To carry out an epigenome-wide association study of DS-ALL, using DNA methylation data from Illumina MethylationEPIC arrays in whole blood from neonates, to identify single CpGs and differentially methylated regions (DMRs) associated with risk of ALL in children with DS. Aim 2: To compare neonatal proportions of white blood cell types, estimated using deconvolution of DNA methylation array data, between DS-ALL cases and DS controls. Aim 3: To explore whether variation in neonatal levels of biomarkers of immune development are associated with risk of DS-ALL. These aims are highly relevant to the new NIH-wide initiative Investigation of Co-occurring conditions across the Lifespan to Understand Down syndromE (INCLUDE) Project, as they will shed light on the etiology of a condition, ALL, that occurs much more commonly in children with DS than in the non-DS population. This proposal will address Component 1 of the INCLUDE Project objective, i.e. to carry out targeted, high risk-high reward research related to Down syndrome, in particular related to immune system dysregulation. This proposal will also address Component 2 as it will capture a molecular snapshot of DS through a cohort study, in this case including DSALL cases and controls from the population based California Biobank and the Michigan Biobank. Moreover, this project is extremely well-suited to address the NCI priorities related to INCLUDE, i.e. investigating ?epigenetic and epidemiologic factors associated with trisomy 21 that lead to greatly increased risk of childhood leukemias.? In addition, Dr. de Smith already received funding to carry out a study examining the role of heritable genetic variation in DS-ALL risk. Together, our studies will comprehensively investigate the etiology of ALL in children with DS. The parent grant of this supplement application, ?Perinatal immune development and risk of childhood acute lymphoblastic leukemia? (R01CA175737), is studying the effects of the mother?s immune status during pregnancy on the child?s immune system at birth, and subsequent risk of developing ALL. The study has funded cytokine assay experiments in 137 non-DS ALL cases and 500 controls, for which genome-wide DNA methylation data from Illumina EPIC arrays is also available (the latter created through funds from Project 3 from P01ES018172, Wiemels PI). This methylation data will be used to explore the effects of the maternal immune system on neonatal blood cell proportions in the child, estimated using well-established deconvolution methods. Therefore, the aims of this supplement mesh well to the scope of the parent award. Performing case-control studies in children with DS, who inherently have a heightened risk of leukemia, may increase our power to discover novel risk factors for ALL, and the significant associations discovered in DS children may be pertinent for ALL risk in children without Down syndrome. Given our extensive research program in childhood ALL epidemiology, and our access to large numbers of non-DS ALL cases and controls, we are perfectly positioned to investigate whether risk factors for DS-ALL may also impact ALL risk in the non-DS population. Finally, a subaim of this supplement is to compare results from our DS control group with non-DS healthy controls, which will provide essential data on the effects of trisomy 21 on neonatal DNA methylation genome-wide and on neonatal levels of immune biomarkers. These studies have not been performed previously on the scale proposed in this study, and not in the context of leukemia risk. In addition to elucidating the causes of DS-ALL, results from this study will, therefore, provide valuable insights into the effects of Down syndrome on neonatal immune dysregulation.